Computational protocol: Toxoplasma gondii Superinfection and Virulence during Secondary Infection Correlate with the Exact ROP5/ROP18 Allelic Combination

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Protocol publication

[…] PECs were harvested by injecting 4 ml of PBS plus 3 ml of air into the peritoneal cavity (i.p.) with a 30-gauge needle and shaking and extracting the cells by puncture with a 16-gauge needle. Cells were filtered through 70-µm cell strainer and washed in FACS buffer, 1% FBS–PBS before staining. The spleen or, in the case of T57 mice, the spleen and lymph nodes were dissected and crushed through a cell strainer, pelleted, and incubated in ACK red blood cell (RBC) lysis buffer (NH4Cl at 0.15 M, KHCO3 at 10 mM, EDTA at 0.1 mM) for 5 min at room temperature, washed in FACS buffer, and filtered again through a cell strainer.For FACS analysis, all preparations were done on ice, and cells were blocked in FACS buffer supplemented with 10% normal hamster serum (Jackson ImmunoResearch) and FcBlock anti-CD16/32 (BD Pharmingen) prior to staining with fluorophore-labeled monoclonal antibodies (MAbs). The following MAbs (1:100 staining dilutions) were purchased from eBioscience unless otherwise stated: anti-CD62L eFluor 450 (MEL-14), anti-CD44 eFluor 450 (IM7), anti-KLRG1–fluorescein isothiocyanate (FITC) (2F1), anti-CD127–phycoerythrin (PE) (A7R34), anti-CD19–PE-Cy5 (1D3), anti-CD8α–allophycocyanin (APC) or anti-CD8α–PE-Cy7 (53-6.7), and anti-CD3ε APC-Cy7 (145-2C11; BD Pharmingen). Cells were washed in FACS buffer prior to analysis on an LSR HTS-2 FACS machine (BD) equipped with BD FACSDIVA software.For intracellular staining of cells following in vitro recall infections, 6 × 105 PECs or splenocytes per well (96-well plate) were plated in T cell medium (10% FBS in RPMI 1640 with GlutaMAX, antibiotics, 10 mM HEPES, 1 mM sodium pyruvate [Invitrogen], and 1.75 µl β-mercaptoethanol per 500 ml [MP Biomedicals]). Cells were infected with parasites (multiplicity of infection [MOI], 0.2) for 18 to 21 h, and 3 µg/ml brefeldin A (eBioscience) was added for the last 5 h of infection. Tissue culture plates were placed on ice, and cells were harvested by pipetting, washed with FACS buffer, blocked, and stained for surface markers as described above. Cells were then fixed with BD Cytofix/Cytoperm and permeabilized with BD Perm/Wash solution according to the manufacturer’s suggestions (BD Pharmingen). Cells were stained with MAbs labeled with APC or PE that recognized IFN-γ (XMG1.2) at room temperature for 1 h or overnight at 4°C. Cells were then washed once in BD Perm/Wash and once in FACS buffer prior to FACS analysis. All data were compensated and processed with FlowJo software (Treestar). […]

Pipeline specifications

Software tools BD FACSDiva, FlowJo
Application Flow cytometry
Organisms Mus musculus, Toxoplasma gondii, Homo sapiens
Diseases Infection